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FINAL REPORT

"RESEARCH ON THE ADRENAL CORTICAL HORMONES AND HYALURONIDASE"

'(Juy 1. 1951 to S1

Jeanette C. Opsahl, Ph.D.C.N.H. Long, M4D.: Project DirectorDepartment of Physiological ChemistryYale University School of Medicine

Yale University

Submitted to the Office of Naval Research, harch 12, 1953Subtask No.: OMN: 442

Contract No.: ;/'Nonr-592 (00) NR 123-11+5Previous report submitted January 1, 1952

FINAL REPORT

Condensed digest of related past and present work done under thesponsorship of the Office of Naval Research, relative to thepituitary, placenta, and adrenal-hyaluronidase relationships.

We have investigated the relationships between the various hormones of the

adrenal cortex, the enzyme hyaluronidase, and its substrate hyaluronic acid, with

specific reference to tissue permeability (9-14, 16)t In addition, during the

past four years, this work has been carried out with a view towards its possible

relation to the field of rheumatoid and other collagen disease processes (15, 17,

and data in process of compilation for publication). As the studies progressed,

several problems presented themselves: a. Which fractions of the adrenal cortex

were responsible for the results obtained. b. What was the mechanism or

mechanisms of action involved. c. In what way was the hyaluronic acid metabolism

involved in the arthritides and in other collagen diseases. A review of certain

of these studies was presented in part, in the book "The Adrenal Cortex", 2nd

transactions, J. Nacy Foundation, 1951 (16). The practical implications of these

findings in the field of collagen diseases are obvious, and further investigations

in many phases of these studies will be discussed in other parts of this report.

II

Our experimental observations indicated a specific relationship between

hyaluronidase inhibition and chemical structure of the adrenal cortex steroids.

In a series of in vivo studies, several pure steroids were investigated in an

attempt to define more accurately the structural or hormonal factors that are relat-

ed to the inhibition of the hyaluronidase-enhanced spreading phenomenon (12). The

experimental evidence indicated that inhibition of the spreading reaction by the

adrenal hormones is restricted to steroids that have an oxygen at Carbon 11,

notably Compounds E, F, and to a lesser extent, Compound A. DOCA, the amorphous

fraction, related sex steroids and growth hormone, have consistently been found to

be without effect. In vitro studies have appeared to bear out this conclusion,

-2-

and experimental evidence suggested a direct action of these adrenal hormones on

the enzyme hyaluronidase in vitro (16, and unpublished studies).

III

Related studies on the role of environmental temperature and humidity in

these processes were in part reported, as suggestive that increased environmental

t amperature and humidity play a role in stimulating the adrenals and thus causing

a decrease in the permeability of the ground substance, (10).

IV

Investigations on the adrenal-hyaluronidase relationship in infectionP

pointed out that ith hyaluronidase-containing bacteria, the course of infection

could be inhibited markedly in normal animals as well as in adrenalectomized

animals by the administration of adrenal cortical extracts; whereas in the case

o,. the hyaluronidase-free bacteria and viruses, a protective effect of adrenal

c rtical extract could be demonstrated only in the adrenalectomized animals. With

both types of organisms, replacement therapy in the adrenalectomized animals did

not give as complete protection against infection as was found in the normal

control animals. It was concluded that while the adrenal-hyaluronidise relation-

ship is one of the factors involved in the lowered resistance of animals to

infection, a lack of hyaluronidase inhibition can be only one component of this

complicated deficiency condition (14, and unpublished studies).

V

We have showa that either adrenocorticotrophic hormone or the C-11 oxygenat-

ed steroids inhibited the hyaluronidase-enhanced spreading phenomenon in the

rabbit and in the mouse (9-16). In view of the favorable influence of pregnancy

on certain of the collagen diseases, it was of interest to determine whether

chorionic gonadotrophins had any influence on this phenomenon. This was found

-3-

to be the case (CG), but in the course of these investigations it became apparent

that the inhibition produced by this agent was due, not to the gonad-stimulating

principle, but to a contaminant which exhibited many of the properties previously

associated with ACTH from pituitary tissue (15). Several similarities in the

biologic activity of ACTH, CG, and CG which had been heat-inactivated to destroy

its gonadotrophic activity (HICG) from human pregnancy urine have been shown.

Furthermore, it was demonstrated that CG e±1bits ACTH-like activity which could

not be associated with its gonadotrophic activity. The following lines of

evidence established this similarity: Both chorionic gonadotrophin and heat-

inactivated chorionic gonadotrophin caused a marked inhibition of hyaluronidase-

enhanced spreading in normal and castrate animals of both sexes in the presence

of the adrenals but were without effect in the adrenalectomized animals. This

effect was cualitatively identical to that found with pituitary ACTH. The

p 6sibility that this effect was due to non-specific toxicity or mediation through

the gonads was ruled out by numerous control studies. In the hypophysectomized

animals, both CG, HICG, and ACTH were found to exhibit the same marked degree of

inhibition of hyaluronidase activity. There were two possibilities as to the

source of the ACTH-like activity found in these preparations from human pregnancy

urine. The first, that the process of extraction included such quantities of

pituitary ACTH as may have been excreted in the urine; the second, that the

placenta may normally have formed the active agent, a portion of which was also

excreted in the urine. These investigations opened new lines of investigations,

and suggested the possibility that ACTI! of human origin might be obtained for

clinical use, thus providing another source to supplement the quantities now

available only from animal sources.

VI

Preliminary investigations of placental extracts proved informative from

several standpoints and although the crude methods of isolation that were employed

-4-

did not permit quantitative interpretations of yields or of purity, results of

these studies indicated that significant quantities of ACTH-Iike material are

produced by the placenta (17). Furthermore, evidence was cited indicating the

lack of measureble quantities of the C-I oxygenated steroids in these tissue

extracts.

In some of our studies, a modification of Lyon's procedure for the extrac'

tion of manraotropic hormone from pituitary tissue was employed; however, because

of the high water content of the placental tissue and blood and the likelihood

that the major portion of the ACTH activity might remain in the first acetone

residue normally discarded in obtaining the active fraction (Fraction I), further

extraction of the residue was made and labelled Fraction II. Since Fraction II.

represents material that is normally discarded in the Lyon's procedure for extrac-

tion of pituitary tissue, it was most interesting that Fraction II of the

nacenta consistently showed a greater degree of activity than did Fraction I,

and that more than twice as large a yield of active dry powder was hereby obtained.

When fresh placental tissue was extracted, the fractions were not only extremely

large for the crude procedures employed in extraction, but also possessed ACTH

activity in terms of hyaluronidase inhibition, in dosage ranges comparable to

those for pituitary preparations. Horever, placental tissue that had been frozen

and stored prior to extraction did not yield fractions comparable in activity

or quantity to those obtained with the use of fresh placenta. Thus, a destruction

of ACTH activity was indicated, even though the tissue remained in the frozen

state. (Astwood has observed this liability of ACTH in the isolation of pituitary

tissue.)

Identification of the type of activity found in the various fractions of

placental extracts were based largely upon previous work from this laboratory

which has shoiz the adrenal steroid-hyaluronidase relationship to be a normally

functioning physiological mechanism. Further, that certain of the pharmacologicI

-5-

effects observed following administration of large quantities of these hormones,

are related to their influence on this complex enzyme system. In these investi-

gations ACTH was shown to be a potent inhibitor of the hyaluronidase-enhanced

spreading phenomenon presumably through the release of effective C-11 adrenal

steroids; and evidence of a correlation of the C-I adrenal steroids in inhibiting

the hyaluronidase-enhanced spreading phenomenon, and inhibition of the release

of reducing sugars from hyaluronic acid by inactivation of hyaluronidase in vitro

has been obtained.

The markedly similar hyaluronidase-inhibitory effects produced by the

various placental extract fractions, by the human chorionic gonadotrophins, and by

human heat-inactivated chorionic gonadotrophin preparations, compared favorably

with the effects produced by the standardized conrmercial ACTH preparations from

the pituitary in normal, adrenalectomized, and hypophysectomized mice. The

T'.acental tissue fractions caused a marked inhibition of hyaluronidase in both

normal and hypophysectomized animals, and with the lar;er dosages of crude

powders, this inhibition approximated 1OW0 of added enzyme. In all cases, the

placental extracts were inactive in adrenalectonized animals.

The possibility that the observed activity was due in any way to the

presence of steroids of the nature of cortisone was ruled out by the fact that

enzyme inhibition was not observed in adrenalectomized or hypophysectomized-

adrenalectomized amimals, and by the fact that the placental tissue extracts were

without effect when administered intradermally at the site of hyaluronidase

injection.

The factors of non-specificity were ruled out by the lack of effectiveness

of tissue extracts and serum albumin, and further supporting this conclusion were

the results of numerous studies carried out in this laboratory that have shown

that toxic substances such as formaldehyde, sper 1,.ne, colchicine and bacterial.

as well as other agents, do not e:icit an inhibition of hyaluronidase in the

-6--

normal or adrenalectomized animals. Although not directly related to the estab-

lishment of ACTH activity in the placental extracts, it is of importance in

,tudying mechanism of action, that epinephrine in amounts of 0.02 mg. or less

per mouse did not influence the spreading reaction, and only when doses of 0.05 mng.

-ixre employed was any degree of inhibition noted and then only in animals with

-tact adrenals (unpublished data).

These studies on ACTH-like activity of placental tissue indicated that the

pl..centa contains relatively large amounts of the hormone. In addition, even

t'Vough the preparations were extremely crude, there was a striking lack of toxicity

wth their administration. For example, very large doses of placental extracts

cou.ld be administered to adrenalectomized or hypophysectomized mice without

lciting signs of acute toxicity, while commercial pituitary ACTH preparations

in the same or smaller dosages proved toxic under these conditions. Since the

'ncterial is of human origin and presumably would not cause a foreign protein

reaction when administered to humans, it is possible that placental ACTH would

have an important practical value as there have been an increasing number of

reports of sensitivity to pituitary ACTH prepartions. The observation that ACTH

activity is obtained with placental extracts is in agreement with the previous

studies from this laboratory that human pregnancy urine preparations heated to

destroy gonadotrophic activity, possess ;CTH activity (15), and it is probable

that the placental extracts contained the precursor of these excretory products.

(Note: Venning and others have established evidence that urinary corticoids in-

crease during pregnancy; Dobriner and others have shovn there is a marked increase

in urine content of a variety of steroids following ACTH administration; it would

be of interest and of importance to establish whether a similar urinary steroid

pattern would result following administration of placental extracts in non-pregnant

conditions.)

Inasmuch as the summarized experiments definitely showed that there was an

-7-

adrenal trophic effect, and since epinephrine waa excluded from consideration as

a mediator of this action, it was concluded that the effect in all probability

-vas trophic for the adrenal cortex. It was therefore quite appropriate to speak

of placental ACTH as a substance which causes the adrenal to produce a factor or

factors which inhibit hyaluronidase, but it was not necessary to assume that this

placental ACTH has the same physiological characteristics as does pituitary ACTH.

Furthermore, it was obvious that the observations above recorded, posed a

iniLtitude of questions to be answered by further studies before the true relation-

ship between placental and pituitary ACTH can be understood.

VII

The technique of measuring hyaluronidase inhibition has been developed to

the point where it can be applied on a semi-quantitative basis as an index of

adrenal activity, and as stated, it has been used as the principal type of

evdence for the measurement of ACTH and adrenal steroid activity in these series

of studies. When inhibition of hyaluronidase-enhanced spreading in the

hypophysectomized mouse (or rat) is used as a criterion of ACTH activity, the

placental preparations just referred to, possess roughly the same order of activity

as Armour's pituitary ACTH, LA-I-A, or Wilson's ACTH (glacial acetic acid extrac-

tion); however, using the mobilization of adrenal ascorbic acid, or eosinopenic

response as an index of ACTH activity, there would appear to result a wide

variation in degree of activity depending on dosages, experimental animals employed,

and routes and time intervals of administration of extracts. On the basis of

these assays, it would appear that extremely large amounts must be administered in

order to obtain a significant decrease in adrenal ascorbic acid; and while the

gross impurities of these crude extracts must be considered, nevertheless we have

at the same time noted that large doses of chorionic gonadotrophins also have to

be used in order to obtain adrenal ascorbic acid decreases that are significant.

(Note: ACTH activity from placental tissue has recently been described in a

series of papers by Tarrantino, Pentini, and other Italian workers, and earlier in

an isolated study by Jailer and Knowlton; in these studies, depletion of adrenal

ascorbic acid has been used as the criterion of ACTH activity. However, the

Italian workers have also used liver glycogen deposition, and cholesterol depletion

ts well as other assay procedures as their basis for estimation of ACTH activity

of placental tissues.) Our observations wherein extremely large doses of placental

extract preparations are necessary to produce a consistently significant fall

in. adrenal ascorbic acid, have been noted with placental.prepahatioas extracted

br different methods (acetone-HCl;acetic acid extraction of acidified acetone

"isd powders; acetic acid extraction of fresh placental tissue with, and without

..urther purification with oxy-cellulose). At the same time, the hyaluronidase-

inhibiting activity of these various preparations have remained constant or have

0increased, and no destruction of enzyme inhibitory0activity has been noted,

Preliminary studies have been carried -out ielative*o adrenal weights in

0 j

normal knd hypophysectomized rats and mi ofOirct adinistra-

tion; howefer further ihvestigations are necessary befoe nferpretation of results0 ai 00go

can be made, as isothe caf withp ion.stides+. Nwever it is to be noted0 0 p eo. o e • 0it

that ,repeated administration of placental extracts to both mice, and rats (daily0 0 0 0 00

over a tw o week time interval) ha produced. nq adrenal.atrtpfV,.no symptoms of

toxicity to tke.animals. WA interestfing t'hatsmalldoses of the various. P0 aa 0 0 0 0

placental extzact fpactions will produce marked hyaluro .dase" inhibition ino 0

hypophysectomized rats, *hile at the same time, larger doses will show little

uf 'ect on adrenl ascorbit acid mobilization in similar animals.

0.0 0,0

0

• Further investigaions concerning these as well as other pertinent factors,0 0

0 0

* are to be continued in the near future. 0 a e a0 : 0 3. 0. ." .o ••

0 0 0 0 0 o 0 s0 0 0 0

o o 0 00.0 00"" "o " 0 "" 0 0 0 " .'0. " 0000 000000 0@ *o ** •

,S ' •0ee * ....:4. *0o *.*0

-9-

(Further studies not yet completed)

VIII

Preliminary studies carried out in fractionating placental tissue and

blood separately, using the modification of the Lyon's method, have suggested

"hat the blood fractions contain a relatively minor quantity of ACTH. Further,

-hat when placentas are washed essentially free of blood immediately after delivery,

3uch placentas can then be stored for appreciable periods of time without loss of

*tyaluronidase-inhibiting activity in subsequently extracted powders. It would

rppear that enzymes, or other substances, present in placental blood are responsi-

)le for the marked loss of activity in powders extracted from stored combined

blood and placental tissues.

Partial purification of the active principle of placental fractions has

been attempted using: (1) Astwood's glacial acetic acid method on acetone-dried

placental powders, (2) Oxycellulose In the purification of acidified acetone

powders, (3) Initial extraction of fresh placental tissue and blood with

oxycellulose and, (4) Initial extraction of fresh placental tissue and blood with

lH acetic acid followed by treatment with oxycellulose for varying periods of time.

Using Astwood's glacial acetic acid method on acetcoe-dried placental powders,

the final yields were similar to those reported by Astvrood on pituitary tissue

(10-20%) however, hyaluronidase-inhibition studies on the discarded fractions

showed an equal degree of inhibitory activity as did the more purified powders.

The resultant powders were essentially comparable in degree of activity to those

produced by the modification of the Lyon's method. Studies where oxycellulose was

employed in final steps in extractive procedures, yielded powders exhibiting in-

creased activity over those obtained with glacial acetic acid extraction; further,

.-.-hour dialysis in distilled water of liquid fractions eluted from oxycellulose

(using O.IN HCI) caused no resulting loss of activity. The initial extraction of

fresh placental tissue and blood with oxycellulose, or with 1N acetic acid

10

followed by treatment with oxycellulose, offered no particular advantages in terms

of yield or final activity of placental powders.

Results of preliminary purification procedures may be summarized by stating

that hyaluronidase-inhibitirg activity of placental ACTH is not destroyed by

treatment as outlined above; that hyaluronidase-inhibitory activity is enhanced

by treatment with oxcellulose but not by treatment with acetic acid alone; that

activity is not lost by the dialysis of oxycellulose eluates, but that no marked

degree of purification, beyond that obtained with the modified Lyon's procedure,

appeared to have taken place. Testing of these fractions was carried out using

hypophysectomized, normal, and adrenalectomized mice and rats.

For example: 1 mg. amounts of Armour's ACTH preparation, LA-1-A, routinely

produced a 35-50% inhibition of hyaluronidase; 1 mg. amounts of pituitary ACTH

extracted by the glacial acetic acid method, produced an average 20-30% inhibition

of added hyaluronidase; while Aetwood's purified oxycellulose preparation in 1 mg.

doses resulted in no definite inhibitory response. These substances were

administered intraperitoneally at three hour time intervals prior to hyaluronidase

testing. In contrast, following oxycellulose treatment of placental powders,

0.05 mg. amounts produced an average 50% inhibition; 1.0 mg. doses resulted in an

average 80% inhibition, whereas 2 mg. doses caused essentially 100% inhibition of

added hyaluronidase. (Note: 5 mg. amounts of "Fraction II", extracted by only

acidified-acetone procedures, produced the largest yields, and an average con-

sistent 100% inhibition of added hyaluronidase.)

Studies employing Astwood's oxyeellulose-purified pituitary ACTH preparation

appeared to show quite marked loss of hyaluronidase-inhibiting activity with

increasing purification of pituitary ACTH; similarly, this point has been noted to

a lesser degree with pituitary preparations extracted by the glacial acetic acid

'-thod. These observations are in direct contrast to studies on adrenal ascorbic

icid mobilization where ACTH activity of pituitary powders is enhanced following

- 11 -

purification procedures. It is not possible at this time to evaluate the dis-

crepancies noted between mobilization of adrenal ascorbic acid and hyaluronidase-

inhibitory activity, and further investigations are necessary; too, the possibility

of "permissive" action must be ruled out.

Ix

Preliminary investigations for the presence of groith hormone in placental

tissues has been made, using the Wilhelmi and Fishman method, and the Astwood

glacial acetic acid and oxycellulose method. It would appear on the basis of

growth, body weight, and tibial assays in short term experiments, that growth

hormone is not produced in appreciable quantities in the human placenta.

Results obtained following the administration of purified growth hormone

cupplied us by Drs. Aaben and Astwood), clearly showed that growth hormone in

io-10 mg. doses was without hyaluronidase-inhibitory effect.

x

General considerations on the ACTH-adrenal-hyaluronidase relationships.

Over the past several years, we have studied a variety of preparations for

their influence in inhibiting the hyaluronidase-enhanced spread of India ink, and

their influence on hyaluronic acid metabolism. It has been a consistent observation

that such preparations as either ACTH from the pituitary, or crude placental powders,

bring about a more marked inhibition of hyaluronidase than is produced by com-

parable doses of the effective adrenal steroids Compounds E and F when administered

cystemically. This indicates that stimulation of the adrenals results in a

.)hysiological mixture of hormones that is more effective than would be predicted on

+he basis of individual components, or in the production of inhibitory hormones

-ot thus far studied, i.e., physiological stimulation of the adrenal providing more

rtimal. conditions for such phenomena as hy-aluronidase inhibition than can be

obtained by the adinistration of the knonm effective C-11 steroids. In an attempt

%o clarify these factors, further in vivo studies have been carried out, altering

the dosages, times of absorption, combination of compounds tested, and other

'oariables in experimental conditions. Compound F, while more slowly absorbed was

found to be equally as effective as Compound E when tested following longer time

intervals of absorption, and maximal inhibitory effects are maintained over longer

time intervals. Studies on the "amorphous" fraction in terms of hyaluronidase-

inhibition, indicate no hyaluronidase inhibitory activity to be present, rather

when administered in large dosages, it appears to have effects similar to those

of' DOCA, and causing hyaluronidase-enhanced spreading. Purified growth hormone

does not inhibit the hyaluronidase spreading phenomenon.

Investigations from this laboratory have indicated that the activity of the

-KI oxygenated adrenal steroids is concerned, at least in part, with a direct

inhibition of the enzyme hyaluronidase thus producing alterations in the

permeability of the ground substances. Further experimental establishment of the

actual role of the C-11 oxygenated steroids in the spreading reaction is of con-

siderable importance, in that it underlies the action of physiological stimulation

by primary or stress substances, as well as by pituitary or placental ACTH.

The fact that pituitary ACTH is far more active than placental extracted

powders as measured by adrenal ascorbic acid mobilization, while on the other hand,

these same two preparations are of approximately equal activity in inhibition of

the spreading reaction, clearly points out the necessity of further studies as to

the mode of action of placental ACTH. Measurements of their comparative activity

on adrenal weights and histochemical alterations in the adrenal cortex may provide

:,r-ther clues as to their difference in effects on the adrenal cortex, together

-th studies on liver glycogen deposition and adrenal cholesterol depletion. As

: ited, the question of permissive action mst be investigated.

'he possibility that placental ACTH would have a practical therapeutic value

nay be considered inasmuch as: 1. Adcinistration in large doses, and over

-13 -

repeated time intervals, causes no evident symptoms of toxicity; 2. In that it i

of hu=n origin. it presumably would not cause a foreign protein reaction; 3,

Sinilar to pituitary ACTH, it produces a marked inhibition of hyaluronidase in the

absence of the pituitary but presence of the adrenal; 4. It produces no adrenal

atrophy; 5. As may be seen from the work of Hench in studies on pregnancy, the

work of Tarrantino, Pentini, Granier, and others, it would appear implicated in

the noted improvement in arthritic and other connective tissue disease processes;

6, It is availabe in large quantities.

PUBLICATIONS

1. Configuration of the glutamic acid of adenocarcinoma protein.Arnow. L. Earle, and Jeanette C. OpsahlScience, 1939, 2.O 257.

2. Partial racemization of glutamic acid in boiling hydrochloric acid solutions.Arnow, L. Earle, and Jeanette C. OpsahlJ, Biol. Chem., 1940, l"2 765.

3. Configuration of glutanic acid isolated from proteins of pig and chick embryotissues.Clark, William G., L. Earle Arnow, and Jeanette C. OpsahlProc. Soc. Exper. Biol. & iled., 1940, LL 767.

4 Configuration of glutamic acid isolated from subacute lymphatic leucemic tissueproteins.Arnow, L. Earle, Jeanette C. Opsahl; and Cecil J. WatsonProc. Soc. Exper. Biol.& ±ied., 1940, 3, 766.

Racemization of glutamic acid with heat.Arnow, L. Earle, and Jeanette C,, OpsahlJ. Biol. Chem,, 1940, 134, 649.

6. Attempted characterization of malignant tissue protein with d-amino acid oxidase.Arnow, L. Larle, and Jeanette C. OpsahlScience, 1940, 1, 431.

7. Racerization of glutamic acid with alkalies.Arnow, L. Earle, and Jeanette C. OpsahlScience, 1941, 23, 214.

8. The optical configuration of glutamic acid isolated from casein hydrolysates bysix procedures.Opsahl, Jeanette C., and L. Earle Arnow.J. Am. Chem. Soc., 1942, 6JL, 2035.

9. The influence of hormones from the adrenal cortex on the dermal spread of IndiaInk with and without hyaluronidase.Opsahl, Jeanette C.Yale J. Biol. & Med., 1949, _ 255.

lO Elevated environmental temperature: Its possible influence on the action ofspreading factor.Opsahl, Jeanette C.Yale J. Biol. & Ked., 1949, 21 433.

- 14-

- 15 -

Ii. Dermal spreading of India Ink with and without hyaluronidase as influenced byhormones from the adrenal cortex.Opsahl, Jeanette C.Yale J. Biol. & Med., 1949, 21, 487.

12. The role of certain steroids in the adrenal-hyaluronidase relationship.Opsahl, Jeanette C.Yale J. Biol. & Led., 1949, U, 115.

13. The effect of adrenocortic!al hormone on the dermal spreading of India Ink innormal and in adrenalectomized mice.

Opsahl, Jeanette C., White A,, and F. Duran-Reynals.Annals New York Acad. Sci., 1950, 52, 1061.

14, The adrenal-hyaluronidase relationship in infection.Opsahl, Jeanette C., and George H. SmithYale J. Biolo & lied., 1951, 23, 361.

i5o Chorionic gonadotropin, ACTH and the adrenal hyaluronidase relationship.Opsahl, Jeanette C., C. N. H. Long, and Ldith G. FryYale J. Biol.& 1ed., 1951, 2, 399.

16. Hyaluronidase and the adrenal cortical hormones.Opsahl, Jeanette C"The Adrenal Cortex" Transactions of the Second Conference, November 1950,

Josiah Macy, Jr. Foundation, New York, 1951.

17. The identification of ACTH from placental tissues.cpsahl, Jeanette C., and C. 1'. Ii. LongYale J. Biol. & Led., 1951, 24, 199.

18. adrenocorticotropic ictivity of Placentel Extracts.EditorialJ.A.M.A., 1952, ,8. April 5, 1226.

I